The superfamily of neurotransmitter-gated receptor channels responsible for fast synaptic transmission plays a crucial role in inter- and intra-cellular communication. These receptors, including glycine, GABA-A, nicotinic acetylcholine, and 5-HT3 receptors, are essential for various cellular and physiological functions and are the potential targets of many pharmacological and toxicological agents. Because of their membrane association, however, these proteins are refractory to high-resolution structural analyses. The gap is now rapidly widening between the abundance of their sequence-based functional characterizations and the lack of high-resolution structural and dynamical information. This project will focus on a representative member of this important superfamily of receptors and study the transmembrane domain structures of human glycine receptor (GlyR) alpha-1 subunit at atomic resolution. The current consensus of the transmembrane channel architecture is an oligomer of 5 subunits, each of which has four transmembrane domains, TM1-TM4. By combining the new protein expression systems, the segmental isotopic labeling, and the state-of-the art high-resolution and solid-state NMR in membrane-mimetic micelles and lipid bilayers, an integrated dissecting-rebuilding approach will be employed to determine the transmembrane domain structures of GlyR with progressively increasing complexity. The central hypothesis is that the secondary and tertiary structures of the transmembrane domains are largely governed by the membranous environment and by the domain-domain interfacial side-chain interactions. Substantive preliminary results have been obtained by the Principal Investigator and collaborators to support the following four specific aims: 1. To engineer, over-express, and purify functional TM2, TMI+TM2, TM2+TM3, and TMI+TM2+TM3 segments of GlyR alpha1 subunit for structural and dynamical measurements by high-resolution NMR; 2. To devise strategies for segmental isotopic labeling of TMI+TM2+TM3 and TM4 with an artificial linker between TM3 and TM4, so that individually labeled (NMR visible) domain can be studied in the context of the whole unlabeled (NMR-invisible) TM assembly; 3. To use high-resolution NMR for structure and dynamics characterization of the transmembrane domains in membrane-mimetic environments; and 4. To determine the spatial orientation of individual TM segments relative to each other and to the membrane by solid-state NMR. The feasibility of every aspect of the proposed studies has been test and proven. With the very recent availability of a 4-A resolution EM structure of the related nicotinic acetylcholine receptor, the present study will generate structural data with atomic resolution that can serve as templates for the future exploration and prediction of the transmembrane channel architecture for GlyR and other receptors in the same superfamily, providing a high-resolution structural basis for future rational design of new therapeutic agents that are highly specific for the diseases related to these receptors.